1. Field of the Invention
The present invention relates to a Bacillus cell for improved production of a polypeptide of interest and a process for producing a polypeptide of interest.
2. Description of the Related Art
Bacillus cells have been widely used for industrial production of polypeptides of interest. Kunst et al., Nature 390: 249-256 (1997) describes the complete genome sequence of the Gram-positive bacterium Bacillus subtilis. 
The problem to be solved by the present invention is to provide a Bacillus cell capable of producing increased yields of a polypeptide of interest.
The solution is based on that the present inventors have identified that a Bacillus cell expressing less than wild-type level of a gene comprising the DNA sequence shown in SEQ ID NO:1 produces increased yields of a polypeptide of interest.
The gene is identified in a Bacillus subtilis cell and is termed yjbH in the Bacillus subtilis cell. See Kunst et al., Nature 390:249-256 (1997).
Further, the inventors have identified a homologous yjbH gene in a B. licheniformis cell (SEQ ID NO:3).
As stated above (see Background; Kunst et al., Nature 390: 249-256 (1997)) the yjbH gene was, at the priority date of the present invention, NOT annotated, i.e. no known function had been associated with the gene. Based on the teaching provided herein it is among the skilled persons"" general knowledge to identify a homologous gene in another Bacillus cell, e.g. by DNA sequence homology to the yjbH genes disclosed herein (SEQ ID NO:1 and SEQ ID NO:3) and thereby readily be able to produce a Bacillus cell capable of producing an increased yield of a polypeptide of interest according to the solution outlined above.
Further, the DNA sequence shown in SEQ ID NO:1 has very low identity to any other known DNA sequences from any cell. A homology search performed in the publicly known databases such as EMBL, showed that no other sequence had any close identity to the DNA sequence shown in SEQ ID NO:1.
Likewise a homology search in the publicly available SWISSPROT database, using the polypeptide sequence shown in SEQ ID NO:2, showed no other polypeptide with any close identity.
Accordingly, in a first aspect the present invention relates to a Bacillus cell for improved production of a polypeptide of interest, wherein the Bacillus cell expresses less than wild-type levels of a gene that comprises:
(a) the DNA sequence shown in positions 1 to 828 in SEQ ID NO:1;
(b) a DNA sequence which is at least 70% identical to the DNA sequence of item (a);
(c) a DNA sequence which encodes a polypeptide sequence shown in positions 1 to 275 in SEQ ID NO:2; or
(d) a DNA sequence which encodes a polypeptide sequence which is at least 70% identical to the polypeptide sequence shown in positions 1 to 275 in SEQ ID NO:2.
Further, the present inventors have identified that a partial sequence shown as DNA sequence from position 1 to 147 in SEQ ID NO:1 is highly conserved.
Accordingly, in a second aspect the present invention relates to a Bacillus cell for improved production of a polypeptide of interest, wherein the Bacillus cell expresses smaller than wild-type amounts of a gene that comprises:
(a) the DNA sequence shown in positions 1 to 147 in SEQ ID NO:1;
(b) a DNA sequence which is at least 70% identical to the DNA sequence of item (a);
(c) a DNA sequence which encodes a polypeptide sequence shown in positions 1 to 49 in SEQ ID NO:2; or
(d) a DNA sequence which encodes a polypeptide sequence which is at least 70% identical to the polypeptide sequence shown in positions 1 to 49 in SEQ ID NO:2.
As it is clear from the above a Bacillus cell as described herein is highly suitable for the production of a polypeptide of interest.
Accordingly, a third aspect of the present invention is a process for producing a polypeptide of interest comprising the following steps:
(i) cultivating a Bacillus of the first or second preceding aspects under conditions permitting production of the polypeptide of interest;
(ii) isolating the polypeptide of interest.
Definitions
Prior to a discussion of the detailed embodiments of the invention is provided a definition of specific terms related to the main aspects of the invention.
The term xe2x80x9ca genexe2x80x9d denotes herein a gene (a DNA sequence) which is capable of being expressed into a polypeptide within said cell. Accordingly, this gene will be defined as an open reading frame starting from a start codon (normally xe2x80x9cATGxe2x80x9d, xe2x80x9cGTGxe2x80x9d, or xe2x80x9cTTGxe2x80x9d) and ending at a stop codon (normally xe2x80x9cTAAxe2x80x9d, xe2x80x9cTAGxe2x80x9d or xe2x80x9cTGAxe2x80x9d)).
In order to express the gene there must be elements, as known in the art, in connection with the gene, necessary for expression of the gene within the cell. Such standard elements may include a promoter, a ribosomal binding site, a termination sequence, and may be other elements as known in the art.
The term xe2x80x9cthe Bacillus cell expresses less than wild-type levels of a genexe2x80x9d according to the first and second aspects of the invention denotes any alterations of the wild-type cell giving rise to a cell which expresses less than wild-type levels of a gene. These alterations may be alterations of a promoter and/or an open reading frame such as deletions, insertions, frame shifts or any manipulations of the DNA as known in the art.
The expression level of a gene in a Bacillus cell altered according to the above is preferably determined by comparing production levels of the polypeptide of interest in said cell with the production level of the polypeptide of interest in the parent non-altered Bacillus cell. If the altered cell produces more of the polypeptide of interest when compared to the non-altered cell, then the Bacillus cell, according to the first and second aspects of the invention, expresses less than wild-type levels of a gene. The actual assay for the determination of production levels of the polypeptide of interest will depend on the specific polypeptide of interest. It is among the skilled persons"" general knowledge to choose the appropriate assay.
Identity of DNA Sequences
The DNA sequence identity in relation to the terms xe2x80x9ca DNA sequence which is at least 70% identical to the DNA sequence shown in positions 1-828 of SEQ ID NO:1xe2x80x9d of the first aspect and xe2x80x9ca DNA sequence which is at least 70% identical to the DNA sequence shown in positions 1-147 of SEQ ID NO:1xe2x80x9d of the second aspect of the invention is determined as the degree of identity between two sequences indicating a derivation of the first sequence from the second. The identity may suitably be determined by means of computer programs known in the art, such as GAP provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wis., USA 53711)(Needleman, S. B. and Wunsch, C. D., (1970), Journal of Molecular Biology, 48, 443-453). Using GAP with the following settings for DNA sequence comparison: GAP creation penalty of 5.0 and GAP extension penalty of 0.3, the analogous DNA sequences referred to above exhibits a degree of identity preferably of at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 97% with the DNA sequence shown in positions 1-828 of the first aspect of the invention; or the DNA sequence shown in positions 1-147 of SEQ ID NO:1 of the second aspect of the invention.
Identity of Polypeptide Sequences
The polypeptide sequence identity in relation to the terms xe2x80x9ca DNA sequence which encodes a polypeptide sequence which is at least 70% identical to the polypeptide sequence shown in positions 1 to 275 in SEQ ID NO:2xe2x80x9d of the first aspect and
xe2x80x9ca DNA sequence which encodes a polypeptide sequence which is at least 70% identical to the polypeptide sequence shown in positions 1 to 49 in SEQ ID NO:2xe2x80x9d of the second aspect of the invention is determined as the degree of identity between two sequences indicating a derivation of the first sequence from the second. The homology may suitably be determined by means of computer programs known in the art such as GAP provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wis., USA 53711) (Needleman, S. B. and Wunsch, C. D., (1970), Journal of Molecular Biology, 48, 443-453). Using GAP with the following settings for polypeptide sequence comparison: GAP creation penalty of 3.0 and GAP extension penalty of 0.1, the polypeptide encoded by an analogous DNA sequence of the invention exhibits a degree of identity preferably of at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, and especially at least 97% with the polypeptide sequence shown in positions 1 to 275 of SEQ ID NO:2 of the first aspect, or with the polypeptide sequence shown in positions 1 to 49 of SEQ ID NO:2 of the second aspect of the invention.
Embodiment(s) of the present invention is described below, by way of example(s) only.
A Bacillus Cell for Improved Production of a Polypeptide According to the First or Second Aspect of the Invention
The gene as described herein is preferably situated between the two well-known genetic markers in Bacillus, srfAA and thyA.
The term xe2x80x9csituated between the two well-known genetic markers in Bacillus, srfAA and thyAxe2x80x9d denotes herein that the gene is found between the 3xe2x80x2-end of srfAA which is situated at approx. 32xc2x0 on the B. subtilis chromosome, and the 5xe2x80x2-end of thyA which is situated at approx. 162.5xc2x0 on the B. subtilis chromosome (F. Kunst et al., 1997, Nature 390(20): 249-256).
The genetic marker srfAA encodes surfactin synthase sub-unit I in Bacillus, reference is made to (Fuma, S. et al., Nucleic Acids Res. 1993; 21(1): 93-7) for further details.
The genetic marker thyA encodes thymidylate synthases A in Bacillus, reference is made to (Tam, N. H. et al., Mol Gen Genet. 1998; 258(4): 427-30) for further details.
The srfAA marker is a member of a superfamily of homologous markers that are found in numerous different prokaryotic cells, such as swrA in Serratia liquefaciens (Lindum et al., J Bacteriol. 1998; 180(23): 6384-8) and grsA in Bacillus brevis (Krause, M. et al., J Bacteriol. 1988; 170(10): 4669-74).
The thyA marker is well known in numerous different prokaryotic cells, such as Neisseria gonorrhoeae (Carlson, J. H. et al., FEMS Microbiol Lett. 1997; 151(2): 225-30), Bacillus amyloliquefaciens (Tam, N. H. et al., Mol Gen Genet. 1998; 258(4): 427-30), Mycobacterium tuberculosis (Cole, S. T. et al., Nature. 1998; 393(6685): 537-44).
The gene as described herein is more preferably situated between the two well-known genetic markers in Bacillus, mecA and tenA (F. Kunst et al., 1997, Nature 390(20):249-256).
The term xe2x80x9csituated between the two well-known genetic markers in Bacillus, mecA and tenAxe2x80x9d denotes herein that the gene is found between the 3xe2x80x2-end of mecA which is situated at approx. 105xc2x0 on the B. subtilis chromosome, and the 5xe2x80x2-end of tenA which is situated at approx. 106xc2x0 on the B. subtilis chromosome (F. Kunst et al., 1997, Nature 390(20):249-256).
The genetic marker mecA encodes a negative regulator of genetic competence in Bacillus, reference is made to (Kong, L. et al., Mol Microbiol 1993 July; 9(2): 365-73) for further details.
The genetic marker tenA encodes a polypeptide which regulates production of extracellular enzymes in Bacillus, reference is made to (Pang, A S. et al., J. Bacteriol 1991 January;173(1):46-54) for further details.
These two markers are well known in numerous different prokaryotic cells, such as Bacillus firmus (Guo, D. et al., Biochim Biophys Acta Jan. 5, 1998; 1389(1): 34-42), Staphylococcus aureus and S. epidermis (Ryffel, et al., Gene. Sep. 28, 1990; 94(1): 137-8), Helicobacter pylori (Tomb, J F. et al., Nature Aug. 7, 1997; 388(6642):539-47).
Among other based on above-mentioned references it is among the skilled persons"" general knowledge to identify these genetic marker genes in a particular Bacillus cell of interest. For example, the specific Bacillus cell may be any Bacillus cell, such as a Bacillus lentus, Bacillus alkalophilus, Bacillus clausii, Bacillus circulans, Bacillus firmus, and a Bacillus thuringiensis cell.
Preferably, it is a Bacillus licheniformis, Bacillus subtilis, or a Bacillus amyloliquefaciens cell.
A preferred embodiment of the invention relates to a Bacillus cell as described herein, wherein the Bacillus cell expresses less than wild-type levels of a gene as described herein due to the gene being inactivated.
The gene may be inactivated according to any of the strategies well known to the skilled person, such as deletions, insertions of frame shift mutations within the gene or in the promoter.
A further embodiment relates to a Bacillus cell as described herein, wherein the polypeptide of interest is an enzyme, such as a protease, a cellulase, a lipase, a xylanase, a phospholipase, or preferably an amylase.
In SEQ ID NO:3 and SEQ ID NO:4 is respectively shown the partial DNA and polypeptide sequences of a yjbH gene from Bacillus licheniformis. The DNA sequences shown in positions 1 to 147 in SEQ ID NO:1 and the DNA sequence shown in positions 1 to 147 in SEQ ID NO:3 are 76% identical. The polypeptide sequences shown in positions 1 to 49 in SEQ ID NO:2 and the polypeptide sequence shown in positions 1 to 49 in SEQ ID NO:4 are 79% identical.
Accordingly these sequences are within the scopes of the first and second aspects of the invention and an embodiment of the invention then relates to a Bacillus cell wherein the gene comprises the DNA sequence shown in positions 1 to 147 in SEQ ID NO:3 or the gene comprises a DNA sequence encoding the polypeptide sequence shown in positions 1 to 49 in SEQ ID NO:4.
In an even further aspect the present invention relates to a Bacillus cell for improved production of a polypeptide of interest wherein the Bacillus cell expresses smaller than wild-type amounts of a gene that comprises:
(a) the DNA sequence shown in positions 1 to 147 in SEQ ID NO:3;
(b) a DNA sequence which is at least 70% identical to the DNA sequence of item (a);
(c) a DNA sequence which encodes a polypeptide sequence shown in positions 1 to 49 in SEQ ID NO:4; or
(d) a DNA sequence which encodes a polypeptide sequence which is at least 70% identical to the polypeptide sequence shown in positions 1 to 49 in SEQ ID NO:4.
All embodiments relating to the first and second aspects of the invention, e.g. preferred homology identities and the situation of the gene between preferred markers are also preferred embodiments in relation to the aspects mentioned immediately above.
A Process for Producing a Polypeptide of Interest Comprising According to the Third Aspect of the Invention
An essential element in this process is the use of a Bacillus cell as described herein.
The specific cultivation strategy conditions permitting production of the polypeptide of interest may be any of the numerous cultivation protocols known to the skilled person.
Similarly, the specific strategy for isolating the polypeptide of interest of item ii) of the third aspect may be any of the numerous isolation protocols known to the skilled person.
Further, any range or device value given herein may be extended or altered without losing the effects sought, as will be apparent to the skilled person for an understanding of the teachings herein.